Use of a vector expressing DNA polymerase β as medicine

ABSTRACT

The invention concerns the use of a vector expressing DNA polymerase β for the treatment of cancer or viral diseases such as AIDS.

BACKGROUND OF THE INVENTION

The present invention relates to the use of a vector which expresses DNApolymerase β as a DNA medicinal product in the context of the moleculartreatment of cancer and viral diseases such as AIDS.

Essentially two types of genetic treatment exist for these diseases,namely immunotherapy and the introduction of “suicide” genes by viralvectors into the target cells.

Genetic immunotherapy is the use of tumor-infiltrating lymphocytes(TILs), which are highly tumoricidal. When they are transformed, theyconvey interleukin (IL2, IL4, IL6, γ-interferon) genes to the tumor andthese activate the immune response locally, allowing both a limitationof the side effects on the body and an amplification of their antitumoreffect.

The approach directed toward transferring a “medicinal DNA” ortherapeutic gene into a tumor cell has already been proposed (Culver etal., 1994) in the context of the use of genes such as the HSV tk gene ofthymidine kinase from the human herpesvirus HSV-1 (Moolten et al., 1990;Culver et al., 1992) or VZV tk if it is from the chickenpox virus (Huberet al., 1991), the bacterial genes gpt coding for xanthine/guaninephosphoribosyl-transferase (Mroz et al., 1993) or codA coding forcytosine deaminase (Mullen et al., 1992). The products of these“suicide” genes convert initially nontoxic agents, such as ganciclovir(GCV) in the case of HSV-TK, 6-methoxypurine (ara-M) with VZV-TK,5-fluorocytosine (5-FC) with CodA and 6-thioxanthine (6-TX) with GPT,into products which are highly toxic to the cell.

Viral thymidine kinase (TK) genes have been used in particular todestroy several types of cancer cells (Moolten et al., 1990; Huber etal., 1991; Vile et al., 1993), making these cells sensitive to purine orpyrimidine analogs such as acyclovir (ACV), ganciclovir (GCV) orbromovinyldeoxyuridine (BVDU). These nucleoside analogs are converted bythe viral TKs into diphosphorylated forms which are thentriphosphorylated with endogenous cell enzymes before incorporation intothe tumoral DNA by DNA polymerases. The incorporation of these chainterminators (absence of 3′ OH) blocks the replication of the DNA andleads to cell death.

DETAILED DESCRIPTION OF INVENTION

The present invention proposes to use a novel type of suicide gene whosefunction consists in facilitating the incorporation of a nucleotideanalog into the DNA of the target cell after phosphorylation of thenucleoside prodrug optionally by a “standard” suicide gene.

The incorporation of nucleotides into DNA is naturally carried out ineukaryotic cells by DNA polymerases. Among the mammalian DNApolymerases, DNA polymerase β has a number of specific features.

DNA polymerase β is a polypeptide of 39 kD and is an enzyme which ishighly conserved in higher eukaryotes (Kornberg et al., 1992). Itsprimary function is believed to be the repair of damaged DNA (Sobol etal., 1996), but it also has a role in the replication of native DNA(Jenkins et al., 1992; Sweasy et al., 1992). DNA polymerase β isexpressed at a constant level during the cell cycle (Zmudzka et al.,1988) and exposure of the cell to xenobiotic agents such as radiationsinduces its expression (Srivastava et al., 1995; Fornace et al., 1989).It differs from the other polymerases in its small size and itsunfaithful nature during DNA replication, this infidelity being linkedto the absence of associated corrective exonuclease activities (Kunkelet al., 1986).

in vitro, it has been shown that DNA polymerase β incorporates ddCMP(triphosphorylated dideoxycytidine), an inhibitor of DNA synthesis, withan efficacy which is comparable to that observed for the incorporationof the natural antagonist dCMP or deoxycytidine monophosphate (Copelandet al., 1992). Very similar results have been published in relation toAZT (Copeland et al., 1992; Parker et al., 1991). in vivo, AZT-MP(azidothymidine monophosphate) is in fact incorporated into cellular DNA(Sommadossi et al., 1989) and it has been suggested that DNA polymeraseβ plays a role in this process (Parker et al., 1991).

Thus, a subject of the present invention is, in particular, the use of avector which expresses DNA polymerase β or an analog of DNA polymerase βin order to incorporate nucleotide analogs with antiviral or antitumoractivity into a cell's DNA, for the manufacture of a medicinal productintended for the treatment of cancer or a viral disease such as AIDS.

The expression “DNA polymerase β analog” means any nucleotide sequencewhich has at least 80% homology with the nucleotide sequence ofmammalian DNA polymerase β and which fulfils the same functions.

The present invention thus consists in inducing an intracellularoverproduction of an enzyme which is normally present in low amount inthe cell, namely DNA polymerase β, in order to amplify its unfaithfuland mutagenic nature and thus force the incorporation of nucleotideanalogs into the DNA.

A subject of the present invention is also an expression vectorcomprising a gene coding for DNA polymerase β or an analog of DNApolymerase β. The vector is intended to express DNA polymerase β intumor cells or cells infected with a virus such as the AIDS virus. It isthus advantageous to place said gene under the control of an expressionsystem which is effective in the target cells.

According to one advantageous embodiment of the present invention, theexpression vector in accordance with the present invention comprises atarget sequence and/or expression sequence which is specific for tissuesin which it is desired to express the DNA polymerase β (or an analog),such as tumors or tissues infected with viruses; by way of example,mention may be made of hematopoietic strain cells for the eradication ofCD4⁺ lymphocytes infected with the HIV virus.

The expression vector according to the present invention can be anyvector commonly used in gene therapy, and particularly a viral vectorderived from a virus chosen from adenoviruses, adeno-associated viruses,retroviruses (including HIV), herpesviruses, poxviruses, parvoviruses,plasmoviruses, Semliki Forest viruses and Sindbis viruses.

As indicated above, DNA polymerase β allows the incorporation ofnucleotide analogs into DNA. Now, certain nucleoside analogs are knownto have antiviral activity when they are phosphorylated, i.e. in theform of nucleotides. This is the case in particular for AZT and ddC.These nucleoside analogs are normally atoxic to cells. However, afterphosphorylation and in the presence of DNA polymerase β (or analog),they are incorporated into DNA and block its replication. Thephosphorylation in question can be carried out in particular bythymidine kinase and/or thymidilate kinase.

Thus, according to one particularly advantageous embodiment, the use ofa vector which expresses DNA polymerase β (or analog) in accordance withthe present invention can be potentiated by the expression, in thesesame target cells or tissues, of the thymidine kinase and/or thymidilatekinase gene.

The cells in which the DNA polymerase β and the thymidine kinase and/orthymidilate kinase are expressed are thus made more sensitive tonucleoside analogs such as AZT or ddC, which, in situ, arephosphorylated before being incorporated into the DNA.

The thymidine kinase and/or thymidilate kinase gene can be inserted onthe same vector as the one which expresses DNA polymerase β or onanother vector. In the latter case, the target cell will undergo aco-transfection in order to allow the expression of each of the genesconcerned. In any case, the gene coding for thymidine kinase and/orthymidilate kinase will be placed under the control of an expressionsystem which is effective in the target cells to be reached.

The subject of the present, invention is also cells transformed with anexpression vector in accordance with the invention comprising a genecoding for DNA polymerase β (or analog) and optionally also comprising agene coding for thymidine kinase and/or thymidilate kinase, as well asto the cells transformed with an expression vector in accordance withthe invention comprising the two abovementioned genes.

Moreover, it should be pointed out that the vectors in accordance withthe present invention can be used as cloning vectors. The insertion of apolylinker into the gene coding for DNA polymerase β (or an analog)without modifying the reading frame, which is itself inserted into avector such as pUT-pol β or pZHTk β, allows a gene of interest to becloned. The recombinant cells transfected with this type of vector canreadily be selected since they have become resistant to nucleosideanalogs such as AZT or ddC following inactivation of the DNA polymeraseβ (or analog) gene.

Lastly, a subject of the present invention is a product containing anexpression vector according to the invention comprising a gene codingfor DNA polymerase β (or analog) and a gene coding for thymidine kinaseand/or thymidilate kinase or two expression vectors each comprising oneof the genes in question and at least one antiviral agent, as acombination product for simultaneous or separate use or for use spreadout over time. Preferably, the antiviral agent is AZT or ddC.

It should also be noted that the use of expression vectors and/or cellstransformed with these expression vectors, in accordance with thepresent invention in the context of the treatment of cancers or viraldiseases such as AIDS, can be combined with any other standard treatmentsuch as chemotherapy or radiotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the construction of the vector pUT-pol β from thevector pUT 687.

FIG. 2 represents the percentage of survival of E.coli SC 18-12 cellstransformed with the vector pUT-pol β at different temperatures, in thepresence of AZT.

FIG. 3 represents the construction of the vector pZHTk β from the vectorpZEOSGO.

FIG. 4 illustrates the comparison, as a percentage of survival, of B16cells transfected with the vector pUT-pol β or with a control vector pUT526 Δ in the presence of ddC.

FIG. 5 illustrates the comparison, as a percentage of survival, of B16cells transfected with the vector pUT-pol β or not with the vectorpUT-pol β in the presence of AZT.

FIG. 6 illustrates the comparison, as a percentage of survival, of B16cells transfected with a vector which expresses DNA polymerase β, with avector which expresses thymidine kinase and thymidilate kinaseactivities or with a vector which expresses both.

The present invention is not limited to the abovementioned description,but, rather, encompasses all the variants; furthermore, it will beunderstood more clearly in the light of the examples below, which aregiven purely for illustrative purposes.

EXAMPLES EXAMPLE 1

Construction of a plasmid vector which overexpresses DNA polymerase β.

Materials:

AZT (Retrovir IV, zidovudine, Azidothymidine) and Ganciclovir (Cymevan)are obtained from the laboratories of Wellcome, Paris and Syntex,Puteaux, S.A., respectively.

Zeocine is produced by the company CAYLA, Toulouse.

The information regarding the E. coli bacterial trains used is collatedin the table below:

Strains Genotypes Sources MC 1061 D(ara leu) aral galU O. Fayet (IBCG,galK hsdS rpsl.Δ(lac University of IOPZYA)X74 ΔrecA Toulouse III) SC18-12 Ion11, sulA 1, trpE65, Witkin (Waksman uvrA 155, fadAB::Tn10,Institute, State recA178, polA12 University, New Jersey

Culture media:

Complete LB medium (per liter): tryptone 10 g, yeast extract 5 g, NaCl10 g, agar 15 g, H₂O qs 1 l.

M9CA minimum medium (per liter): Na₂HPO₄.2H₂O 8.5 g, KH₂PO₄ 3 g, NaCl0.5 g, NH₄Cl 1 g, 0.4% glucose, 0.2% caseine hydrolyzate, agar 1.5 g,H₂O qs 1 l.

NA medium (per liter): “Bacto Nutrient Agar” from Difco, Bacto BeefExtract 3 g, Bacto Peptone 5 g, NaCl 10 g, agar 15 g, H₂O qs 1 l.

Transformation:

Competent cells are prepared according to the Kushner method. 25 ml ofsterile LB are inoculated with the strain and, at OD₆₀₀=0.4, centrifugedfor 15 minutes at 5000 rpm, at 4° C. The cells are taken up in 2.5 ml of0.1 M CaCl₂/10 mM MOPS/0.5% cold glucose left for 30 minutes at 0° C.100 μl of competent cells are incubated with the plasmid DNA (volumeless than 10 μl, 10 to 50 ng per ccc plasmid) for 30 minutes in ice andthen for 5 minutes at 42° C. without stirring. LB medium is then addedat room temperature (qs 1 ml) and the cells are placed at 37° C. (MC1061) or at 30° C. (heat-sensitive SC 18-12) with stirring (NewBrunswick, 300 rpm) for 1 h 30 min for the phenotypic expression, andare then plated out on Amp selective medium (LB+Ampicillin 100 μg/ml)for the vector pUT, or on Zeo elective medium (LB+Zeocine 20 μg/ml) forthe vector TG.

Cloning:

The DNA fragments obtained from the appropriate enzymatic digestions areseparated by electrophoresis on Sea Plaque low-melting agarose gel (FMC)at 0.7% in TAE buffer (0.004 M Tris-acetate/0.001 M EDTA). The bandschosen are cut up, liquefied in TE 1× adjusted to 0.5 M NaCl and themixture is heated for 10 minutes at 70° C. The DNA solutions are thenpurified by extraction with phenol-CHISAM and then precipitated with onevolume of isopropanol+1 μl of glycogen. The pellets are then dried andtaken up in the minimum volume of sterile water (10 μl). The requiredDNA fragments are mixed and incubated in a ligation buffer (66 mMTris-HCl, 5 mM MgCl₂, 1 mM polyethylene glycol, 1 mM ATP, pH 7.5) withone unit of T4 phage ligase (20 μl final) at 16° C. overnight. Theligation product is then used to transform the adequate strain. Theadapted selection is either resistance to 100 g/ml ampicillin or to 20μg/ml Zeocine, depending on the plasmid.

Extraction of the plasmid DNA:

The plasmids are prepared according to the method of Birboim and Doly(Birboim et al., 1979) by alkaline lysis of the bacteria from streaks ondishes or from 25 ml cultures. The plasmid DNA of the transformants isextracted and checked by restriction analysis.

Standard PCR conditions:

The polymerase chain reaction is carried out in 500 μl microtubescontaining 5 μl of 150 μM MgCl₂ buffer, a mixture of the 4 dNTPs (250 μMeach), 10 ng of matrix DNA and 500 ng of each oligonucleotide primer ina reaction volume of 100 μl, to which 2 to 3 drops of oil are added toprevent any evaporation. After 7 minutes of pre-denaturation at 95° C.,2.5 units of Tfl Polymerase enzyme (EPICENTRE) are added “Hot-Start”.The amplification is obtained after 25 cycles of denaturation (94° C., 1min), hybridization (55° C., 1 min) and extension with polymerase (72°C., 1.5 min).

The primers used consist of two parts: a part which hybridizes with oneend of the gene to be cloned and a non-hybridizing part which includes arestriction site which is compatible with the cloning site in theexpression vector. Purification of the PCR product by electrophoresis onSea Plaque low-melting 0.7% agarose gel (TEBU) and controlled byenzymatic digestion. The ends are cleaved and ligated inside thecomplementary sites of the expression plasmid (Enzymes Biolabs andBoehringer).

Rat polymerase beta gene:

The cDNA of purified rat DNA polymerase was generously donated by DrWilson (Galveston, Tex., USA). The sequence of this gene was obtained bythe EMBL European Data Bank.

Vector:

A vector was constructed in which the CDNA of the rat polymerase genewas cloned after amplification by the PCR technique. pUT-Pol β

The plasmid pUT-Pol β results from replacement of the E. coli thymidinekinase gene (fragment NcoI-AvrII) with polymerase β (fragment NcoI-NheI)fused with the Zeocine-resistance gene in the plasmid pUT 687. Thepolymerase β gene is placed under the control of two strong constitutivepromoters in tandem, the bacterial promoter EM7 and the eukaryoticpromoter of the TK gene from HSV with its enhancer, known as the“shuttle” vector, which can be used in E. coli and in eukaryotic cells(FIG. 1).

5′ primer: Addition of 2 CA bases allowing the rat polymerase β gene tobe introduced in the open reading frame. These 2 bases create a codonGCA coding for an Alanine. The site Nco I provides the construct withthe site for initiation of the ATG translation.

3′ primer: loss of the single restriction site Avr II from the plasmidduring Avr II/Nhe I ligation.

Loss also of the “Stop” codon for the polymerase β gene to allow fusionof this gene with the Zeocine-resistance gene, a counterselection of theAZT clones will allow the clones which have lost the E. coli thymidinekinase to be selected. Summary of the construction of the vector pUT-polβ:

Vector constructed PUT - Pol β Parental vector pUT 687 E. coli thymidinekinase::zeo Resistance provided Zeocine (if the fusion is correct)Ampicillin Gene cloned rat polymerase β cDNA 5′ PCR primer 5′ TAT TCC ATG  GCA CTC GTG GAA CTC GCA AACTTT 3′ (SEQ ID NO: 1) Restriction sitesupplied Nco I: CC ATG G GG TAC C 3′ PCR primer 5′ TTA AGC  TAG  CTC ACTCCT GTC CTT GGG CTC 3′ (SEQ ID NO: 2) Restriction site supplied Nhe I:GCT  AGC CGA TCG Ligation 5′ Nco I/Nco I 3′ nhe I/Avr II

Determination of the MIC (minimum inhibitory concentration):

The cells are plated out on solid LB medium overnight. They are taken upin 1 ml of LB medium containing 20% glycerol final and stored at −700°C. 5 μl of a suspension of 0.5 ml of M9CA medium are inoculated with anestimated constant amount of frozen cells taken up with a platinum loopso as to obtain an OD₆₀₀=0.1-0.2, and deposited on M9CA mediumcontaining increasing concentrations of drug. After leaving overnight at37° C., the lethal concentration is noted for the different strains.

Survival tests:

The survival test makes it possible to demonstrate sensitization of thestrain to the drug by DNA polymerase β.

E. coli B/R SC 18-12 bacteria are incubated overnight in 25 ml of liquidNA medium with 35 μg/ml ampicillin, at 30° C. with stirring (NewBrunswick 300 rpm), diluted so as to obtain an OD₆₀₀=0.1-0.2 in 20 ml ofNA medium, and recultured under the same conditions as above until anOD₆₀₀=0.4 is obtained, they are then diluted 10⁵-fold in liquid M9CAmedium, 100 μl are plated out on M9CA dishes containing differentconcentrations of drug, and incubated for 1 to 2 days at 37° C. or at30° C. The number of colonies is counted on each of the dishes, and thepercentage of survival is determined relative to the maximum value ofthe drug-free control dish.

In parallel, RecA polA12 mutant E. coli SC 18-12 strain was transformedwith the vector pUT-Pol β. DNA polymerase I is heat-sensitive at 37° C.,but functions at 30° C. The overexpression of DNA polymerase β, at anon-permissive temperature of 37° C., restores the viability, asexpected.

The plasmids of these bacteria were extracted, digested with Eco RI andthe electrophoretic profiles were checked. We also carried out a controlby PCR with the probes used for the cloning, and the DNA polymerase βgene is indeed borne by plasmids extracted from the test bacteria.

Another bacterial strain MC 1061 was also transformed by the vectorpUT-Pol β, in order to observe a change in the minimum inhibitoryconcentration with AZT and Ganciclovir, mediated by DNA polymerase β.

1—Strain MC 1061/pUT-pol β

With respect to AZT:

The “spots” obtained on the dishes for the bacteria MC 1061-pUT-Pol βshow that, with respect to AZT, the value of the MIC goes from 0.03μg/ml for the parent control strain to 0.001 μg/ml (factor 30), withoutthe presence of the thymidine kinase-HSV1 suicide gene. The AZT is toxicto the bacteria.

The results are collated in Table 1 below.

TABLE 1 Minimum inhibitory concentrations (μg/ml): AZT Azidothymidine MC1061 - TK+ 0.03  non-transformed MC 1061 - TK+ 0.001 pUT - Pol β

2—Strains SC 18-12/pUT-pol β

With respect to AZT (FIG. 2):

The experiment at 37° C. shows a dose/response effect for AZT, asexpected. The MIC falls with the strain SC 18-12/pUT-Pol β by a factorof 3 at 30° C. and by a factor of 30 at 37° C., relative to the parentstrain at 30° C. This confirms the functionality of our vector and showsthat the “suicide” effect of DNA polymerase β in bacterial cells exists.

EXAMPLE 2

CHO or B16 animal cells which overexpress Pol β become sensitive to ddCand to AZT.

Culture media:

The materials are obtained from Biowhittaker. Heraeux oven at 37° C., 5%CO₂.

CHO: the Chinese hamster ovary cells (Line supplied by J.Tessié-IBCG-Toulouse) are cultured in L-glutamine-free α-MEM-HEPESmedium supplemented with calf fetal serum (10% FCS), L-glutamine,penicillin/streptomycin (PS 50 μg/ml).

B16: the mouse melanoma cells (Line B16b16 supplied by S.Cros-I.P.B.S.-Toulouse) are cultured in an RPMI 1640 medium,supplemented with horse serum (10% HS), penicillin/streptomycin (PS 50μg/ml).

Transfection of the CHO/B16 cells:

The technique used for the 2 types of cell is identical; only a fewparameters are different.

A number of confluent cells (CHO=2×10⁵/B16=4×10⁵) are placed in 2 ml ofsupplemented medium. At 24 hours the cells at subconfluence are washedwith PBS and placed in 1 ml of serum-free medium containing 10 μl ofAldrich polybrene (1−/−) and 10 μg of DNA. At 30 hours for the CHO andat 40 hours for the B16, the cells are subjected to a DMSO shock (1 ml30% DMSO for 3-4 minutes), 2 rinses with serum-free medium, incubationfor 72 hours and addition of the Zeocine selection (CHO 100 μg/ml; B16:10 μg/ml).

Recovery of the clones:

About 15 days for the CHO (3 to 4 weeks for the B16) after washing withPBS, the clones are recovered by scraping with a micropipette in 5 μl ofmedium, and are deposited individually or as a “pool” in a dish with adiameter of 35 mm containing 2 ml of medium supplemented with Zeocine(100 or 10 μg/ml).

Determination of the MIC (minimum inhibitory concentration)

The confluent cells are detached with trypsin after rinsing with PBS(phosphate buffer saline/Biowittaker) and are taken up in 1 ml ofsuitable medium with serum and antibiotics.

1 ml of supplemented medium is added to each well (24-well Nunc plate)along with 2000 cells for the CHO or B16, and the various prodrugs ofincreasing concentrations.

A first reading of the MIC values is taken after 5 days for the CHO and7 days for the B16; the medium is replaced with a drug-free medium andthe true MIC is determined at 10 and 14 days, respectively.

Studies of the toxicity of DNA polymerase β overexpressed in eukaryoticCHO and B16 cells:

DNA polymerase β is a constitutive eukaryotic enzyme, known as a“house-keeping” enzyme. A constitutive overexpression of this polymeraseshould not present too large a toxicity for the transfected cells. Thetwo types of cell (CHO and B16) were transformed with the vectors. Inthe two cases, the plasmid introduced cannot be replicated and must beintegrated randomly into the cell genome. For these reasons, a pool of 5clones of CHO was also tested in order to have a precise and overallconfirmation on this type of cell.

The vector pUT-Polβ shows that the overexpression of DNA polymerase βalone is certainly not toxic to the cell during division, since we haveobtained many clones. The most surprising result is that this eukaryoticpolymerase by itself very markedly sensitizes the eukaryotic CHO cellsto AZT (MIC from 10 to 30 μg/ml), with a factor of greater than 30 for 3out of 5 clones.

The results are summarized in Table 2 below.

TABLE 2 Minimum inhibitory concentrations for the CHO cells (μg/ml AZTAzidothymidine Non-transfected/zeo >300 pUT: Polymerase 10/30 β.//zeo

EXAMPLE 3

Construction of an expression vector bearing both polβ and the HSV tkthymidine kinase gene from the human herpesvirus HSV-1 which codes bothfor thymidine kinase activity and thymidilate kinase activity.

The plasmid pZHTkβ (FIG. 3) was constructed by amplifying a fragment ofpUT-pol β containing pol β, by PCR by means of the use of theoligonucleotides:

5′ TTCTCAGTGACCGGCGCCTAGT 3′ (SEQ ID NO:3)

5′ GGGAGCCCAAGGACAGGAGTGAATGATTCGAACTTT3′ (SEQ ID NO:4)

After BbrpI-BstBI cleavage, the PCR fragment was inserted into a vectorpZEOSGO opened with SpeI-BstBI and then treated with Klenow polymeraseat the SpeI end. pZEOSG0 is derived from pZEOSV1 (Cayla, VECT 2001).

The functionality of the construct was checked using E. coli bacterialstrains,

1—either by transforming the strain SC 18-12 for the expression of polβ,

2—or by transforming a strain SC 18-12 tk⁻ which is deficient forthymidine kinase activity, for the HSV TK thymidine kinase expression.The mutant tk⁻ was obtained by plating out SC 18-12 bacteria on a mediumcontaining AZT. Only the cells deficient in TK activity grow on such amedium, since the AZT is not phosphorylated in this case and thus cannotbe incorporated into the DNA,

3—or by transforming the strain TD205, a heat-sensitive mutant forthymidilate kinase activity, for the HSV TK thymidilate kinase activity.

Table 3 below shows that the sensitivities of the strains used tozeocine, to AZT or to a non-permissive temperature are suppressed aftertransformation with the plasmid bearing the hybrid gene HSVtk::Sh andthe cDNA of pol β.

TABLE 3 Survival Zeocine AZT (%) 25 10 E. coli 37° C. 42° C. μg/ml μg/mlSC 18-12 100  5  5 ND SC 18-12/pZHTkβ  90 50 97 ND SC 18-12 tk⁻ ND ND ND100 SC 18-12 tk⁻/ ND ND ND  2 pZHTkβ TD205 100 11 ND ND TD205/pZHTkβ 10078 ND ND

EXAMPLE 4

Sensitivity to ddC of mouse melanoma cells transfected with pUTpolβ

Highly metastatic cancer cells of mouse melanomas, B16 cells, weretransfected with the vector pUT-pol β by the DMSO/polybrene method.

Recombinant cell extracts were prepared and then analyzed by Westernblotting against anti-polβ antibodies. This experiment showed theoverexpression of pol β in the transfected cells. We also showed duringDNA replication tests that these extracts allowed an in vitroincorporation of triphosphorylated ddC into the G-rich nucleotidesubstrate:

3′CATACGAGAACCAACAT 5′ (SEQ ID NO:5)

5′ GGTGGTGGTGGGCGCCGGCGGTGTGAATTCGGCACTGGCCGTCGTATGCTCTTGGTTGTA 3′ (SEQID NO:6)

This result shows the specificity of pol β in the incorporation of ddCinto DNA, leading to blocking of the replication of this DNA.

FIG. 4 shows the toxicity of ddC on B16 cells transfected with pUT-pol βrelative to cells transfected with a control vector pUT526Δ containingno polβ but only the selection gene Sh. The cell survival was measuredby staining the treated cells with Giemsa in order to compatibilizethose cells which survived being placed in contact with the drugs.

EXAMPLE 5

Mouse melanoma cells transfected with pUT-pol β are sensitized to AZT.

Experiments similar to the ones above were carried out using AZT.

FIG. 5 shows the toxic effect of AZT on cells transfected with pUT-polβ.

EXAMPLE 6

Mouse melanoma cells which express HSV TK::Sh and Pol β are moresensitive to AZT than cells which express HSV TK::Sh or Pol βseparately.

B16 cells were transfected with the vector pZHTkβ which co-expresses Polβ and an HSV TK::Sh protein by fusing,

HSV TK expressing the thymidine kinase (TK) and thymidilate kinase (TMK)activities of the herpesvirus HSV-1, i.e. capable of mono- and thendiphosphorylating thymidine and also a wide range of nucleoside analogs,

the protein Sh which confers resistance to zeocine.

FIG. 6 shows that these cells are more sensitive to AZT than cells whichexpress Pol β::Sh or HSV TK::Sh separately.

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6 1 33 DNA Artificial Sequence Description of Artificial Sequence PCRPrimer 1 tattccatgg cactcgtgga actcgcaaac ttt 33 2 30 DNA ArtificialSequence Description of Artificial Sequence PCR Primer 2 ttaagctagctcactcctgt ccttgggctc 30 3 22 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide for PCR 3 ttctcagtga ccggcgccta gt22 4 36 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide for PCR 4 gggagcccaa ggacaggagt gaatgattcg aacttt 36 517 DNA Artificial Sequence Description of Artificial Sequence G-richnucleotide substrate 5 tacaaccaag agcatac 17 6 60 DNA ArtificialSequence Description of Artificial Sequence G-rich nucleotide substrate6 ggtggtggtg ggcgccggcg gtgtgaattc ggcactggcc gtcgtatgct cttggttgta 60

What is claimed is:
 1. An expression vector comprising a polynucleotidesequence encoding DNA polymerase β and a polynucleotide sequenceencoding thymidine kinase or thymidilate kinase, wherein saidpolynucleotide sequences are under the control of an expression systemwhich is effective in expressing said polymerase β in a cell.
 2. Thevector according to claim 1, wherein the vector is a viral vectorderived from a virus selected from the group consisting of anadenovirus, adeno-associated virus, retrovirus, herpesvirus, poxvirus,parvovirus, plasmovirus, Semliki Forest virus, and Sinbis virus.
 3. Thevector of claim 1 which is pZH tk pol β.
 4. An isolated cell which istransformed with the expression vector according to claim
 1. 5. Apharmaceutical composition comprising the expression vector according toclaim 1 and at least one nucleoside analog.
 6. The pharmaceuticalcomposition according to claim 5, wherein said nucleoside analog is AZTor ddC.
 7. A pharmaceutical composition comprising an expression vectorcomprising a polynucleotide sequence encoding DNA polymerase β and atleast one nucleoside analog, wherein said polynucleotide sequence isunder the control of an expression system which is effective inexpressing said DNA polymerase β and induces overproduction of said DNApolymerase β in tumor cells or cells infected by a virus.
 8. Thepharmaceutical composition according to claim 7, further comprising anexpression vector comprising a polynucleotide sequence coding forthymidine kinase or thymidilate kinase which is under the control of anexpression system which is effective for the expression of saidthymidine kinase or thymidiiate kinase in sad cell.
 9. Thepharmaceutical composition according to claim 8, wherein said nucleosideanalog is AZY or ddC.
 10. The pharmaceutical composition according toclaim 7, wherein said nucleoside analog is AZT or ddC.
 11. A method forenhancing the capability of a tumor cell in incorporating a nucleosideanalog having antiviral or antitumor activity into DNA of said cell,comprising introducing directly into said cell with an expressionvector, thereby inducing overproduction of DNA polymerase β in saidcell, wherein said expression vector comprises a polynucleotide sequenceencoding DNA polymerase β under the control of an expression systemwhich is effective in expressing said DNA polymerase β and inducesoverproduction of said DNA polymerase β in said tumor cell.
 12. Themethod according to claim 11, wherein said vector is used in combinationwith a vector expressing thymidine kinase or thymidilate kinase.
 13. Themethod according to claim 11, wherein said vector is used in combinationwith at least one nucleoside analog.